Wednesday, January 13, 2010

Fourth time is the charm: the quest for the final plasmid

ResearchBlogging.orgIn a previous post, I highlighted the wonders of using yeast recombinational cloning (YRC) as an alternative to “classic” cloning, particularly when under a high-throughput approach [See An alternative cloning strategy: yeast recombinational cloning].

Just to refresh your memory, and in a nutshell, the idea is to co-transform the DNA segment to be cloned into yeast along with the linearized target plasmid, provided that this DNA segment bears homology to defined plasmid sequences. By homologous recombination, yeast machinery will directly “ligate” your DNA segment into the linearized vector (See fig.1 in the preceding post).

As I stated in the previous post, my interest was to use YRC to clone promoter sequences upstream of a reporter gene (and I have MANY promoter sequences to clone, so this is a suitable approach), and after discussing the methodology and how it applied to my own research, I ended that post by saying:

(…) we have generated a yeast strain which contains a plasmid with our promoter of interest controlling the reporter gene. Now, it’s time to obtain that plasmid in working concentrations, sequence it and then transform it into our model organism.

How do we do this? How do we get the resulting plasmid out of yeast?
The purpose of this post is to discuss how we do this: how we get our plasmid out of yeast so that we can use it to transform our model organism to study the promoter’s biology in vivo.

The answer? A “Smash & Grab” approach (reflecting that you’d smash yeast cells and get what’s rightfully yours: your plasmid).

When I first got to the lab, my PI gave me a protocol that consisted on picking one of the yeast transformants (a yeast colony that grew on URA- media plate after transformation with the DNA segment of interest and the linearized plasmid), streaking it onto a new selective plate and growing it O/N. The next day, you’d scrap the clone and transfer it to a Eppi containing 300 ul of S&G buffer (which has Triton X-100, SDS, NaCl, Tris pH 8 and EDTA), resuspend, add glass beads and phenol/chloroform and vortex for a minute.
You’d then centrifuge and transfer part of the aqueous solution to a new Eppi containing sodium acetate 3M pH 5.2. Then, after an ethanol precipitation, you would have yeast DNA, both plasmid and chromosomal of origin.
After resuspending the DNA in 50 ul of water or TE, you’d take 3 ul and transform E. coli (remember that the plasmid we are using is a shuttle vector).

Even though this seems pretty easy, the efficiency of such method was disastrous in our hands (we use chemocompetent E. coli). My PI insisted this was routinely used in his previous lab (where he did his post-doc and used overpriced commercial electrocompetents cells) with great results (lots of E. coli colonies), but we just couldn’t get it to work with our cells nor we had those commercial electrocompetent cells available. This drove me to literature in order to find a protocol that would give good results in our lab.

I dug up a paper from 1995 (see ref below1) describing a protocol which was very similar to the one we were using, although it claimed to yield plasmid DNA without chromosomal contamination, which is perfect.

Briefly, two/three loopfuls of yeast cells (taken directly from patches on minimal media, grown O/N), were resuspended on a solution containing NaCl, Tris-HCl pH 8, EDTA and SDS. Then, glass beads were added, and the mixture was vortexed for 2 mins. An ice cold solution containing NaOH and Triton X-100 was then added, and after mixing by inversion, an aliquot of sodium acetate 3M pH 4.8 was added, and everything was then mixed thoroughly. After a short incubation in ice, you’d add an equal volume of phenol chloroform-isoamyl alcohol (25:24:1, v/v), vortex the mixture and then centrifuge. Another phenol-chloroform extraction of the aqueous solution was next. Then, the aqueous solution was ethanol-precipitated.

This actually worked like a charm the first time in my hands, but my colleagues couldn’t get it to work. Afterwards, and due to the “magic” of life in the lab, I couldn’t get it to work either, despite several tries!
I didn’t put much thought into it and instead, looked for another protocol, considering that this thing only worked once, and only for one person. I went back online and found another somewhat related protocol2, which “takes advantage of the finding that phenol/chloroform extraction in the presence of LiCl and Triton X-100 solubilises plasmid DNA, while precipitating cellular proteins and denatured chromosomal DNA”.

No good. Transformation efficiency was still dismal in our hands.

Decided to tackle this problem as soon as possible, and with a little help from Google & Pubmed, I found what is now the standard Smash & Grab protocol in our lab: “A simple and highly efficient procedure for rescuing autonomous plasmids from yeast” (my emphasis), an article published in NAR in 19923. How did I miss this in my first search? This is exactly what I was trying to achieve!

So, how is this protocol different?

[from the article]
In order to take advantage of the extremely powerful techniques of molecular genetics which are available in the yeast Saccharomyces cerevisiae, it is often necessary to recover shuttle vectors replicating in yeast back into E. coli. However, many workers have found this step troublesome due to what has been described as a 'persistent inhibitor of E. coli transformation' which accompanies yeast DNA preparations. None of the previously published protocols completely eliminates this problem.
(my emphasis)

Notably, one of the referenced articles which “did not eliminate the problem”, is the one I used in my third try! So after just reading the first paragraph of this one-page article, I became excited.

Besides its claimed higher efficiency (which again cited the article I used in my third try, stating that it was 1-2 orders of magnitude more efficient), this method doesn’t require organic solvents (phenol and chloroform), which is always appreciated :)

The idea behind this protocol is simple: let’s simply boil yeast!

In the words of Montell Jordan, “this is how we do it”: We grow an O/N 5 ml yeast culture under selective conditions (i.e in URA- minimal media), harvest the cells by centrifugation, resuspend the pellet in a buffer containing sucrose, Tris pH8, EDTA and Triton X-100, add glass beads and vortex vigorously for 5 mins. We then place the tubes in a boiling water bath for 3 mins. After a brief cool-down on ice, we spin the cells and transfer the supernatant to a fresh tube with 7.5 M ammonium acetate and incubate for an hour at -20C. Interestingly, the authors claim that:

At this stage, residual chromosomal DNA, large RNA species, and the putative impurities which inhibit E.coli transformation are precipitated
Then, we centrifuge again and add an aliquot of the supernatant to ice cold ethanol and incubate again for an hour at -20C. Later, we centrifuge, wash with 70% ethanol and resuspend in water. We then use 10ul to transform E. coli.

This has reproducibly given great results in our lab (not only in my hands this time ;-)), so we are happy to have finally nailed down this part of the protocol.

But let’s remember that the idea is to get the plasmid out of yeast in working concentrations, sequence it and later use it to transform our model organism.

Now that we have the correctly assembled plasmid in bacteria (which we check by PCR), the aforementioned steps are simple: we just grow a colony O/N in selective media, do a miniprep and send an aliquot out for sequencing.

And that’s it!

In just a week, you can have your segment of interest cloned in a particular plasmid and send it out for sequencing, thanks to the power of yeast genetics (and its high levels of homologous recombination) and to a simple, cheap and efficient protocol for getting plasmid DNA out of yeast and into E. coli(*).

We are now ready to transform our model organism to study the promoter in vivo.
What reporter do we use? How do we monitor it? Be sure to check the next post on this series of articles. Stay tuned!

(*) There are some “yeast miniprep kits” available, but as expected, they are pricey and, in my opinion, not a justifiable expense considering the protocol I just detailed, which is not only cheap, but also easy and amenable for upscaling.

Sobanski, M., & Dickinson, J. (1995). A simple method for the direct extraction of plasmid DNA from yeast Biotechnology Techniques, 9 (3), 225-230 DOI: 10.1007/BF00157083

Ward AC (1990). Single-step purification of shuttle vectors from yeast for high frequency back-transformation into E. coli. Nucleic acids research, 18 (17) PMID: 2205843

Robzyk K, & Kassir Y (1992). A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic acids research, 20 (14) PMID: 1641351

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Alethea said...

That was an awesome post. I have only worked in E. coli or vertebrates, personally, but I really enjoyed your enthusiasm and the can-do approach. It reminds me of trouble-shooting other long, involved techniques and the joy when you finally get it, and get results. We've become too results-oriented. Sometimes the voyage itself is worth the trip, not just the destination. Thanks for writing this.

Alejandro Montenegro-Montero said...

Thank you so much for your kind words and stay tuned for more MolBio Research Highlights!